Osteoclast formation from human cord blood mononuclear cells co-cultured with mice embryonic metatarsals in the presence of M-CSF

Investigating the potentiality of cord monocytes to differentiate toward osteoclast-like cells (OCL) in vitro, we previously reported that in the presence of 1,25(OH) 2 vitamin D 3 (1,25-(OH) 2D 3), multinucleated-cells generated by cord monocyte cultures though displaying morphological features of OCL failed to resorb devitalized bones. We thus hypothesized that full differentiation of cord monocytes toward bone-resorbing cells may require the presence of factors released from and/or direct interactions with living osteogenic cells. In the present study, we tested these hypotheses using two culture systems supporting the development of bone-resorbing cells in the presence of bone matrix. First, cord mononuclear cells were co-cultured with murine fetal metatarsals depleted of osteoclast progenitor cells (stripped metatarsals) in the presence of 1,25-(OH) 2D 3. We found that cord mononuclear cells failed to differentiate toward OCL as indicated by the absence of the release of 45Ca previously incorporated in fetal bones and by the absence of formation of TRAP-positive (TRAP[+]) multinucleated cells which have invaded mineralized cartilage during the co-culture period. In the same model, we then investigated the effect of some soluble factors known as stimulators of osteoclast differentiation. Whereas exogenous rhIL6 and rhIL3 were ineffective in this assay, rhM-CSF consistently increased both the number of TRAP(+) multinucleated cells inside the mineralized cartilage and the release of 45Ca into the culture media. The effects of rhM-CSF were time-dependent reaching the maximum after 3 weeks of culture. In stripped metatarsal cultures treated with rhM-CSF but in the absence of cord mono-nuclear cells, no TRAP(+) cell was visible inside the mineralized area and the release of 45Ca was unaffected. Finally, in the so-called pit formation assay in which cord mononuclear cells were cultured on slices of devitalized bones, we observed that these cells did not form any resorption pit, even when cultured in the presence of rhM-CSF. We suggest that M-CSF is required for full differentiation of cord mononuclear cells toward OCL, in the presence of living bone.